Project Abstract Bladder cancer (BCA) is a major cause of cancer-related death worldwide, with grim 5-year survival rates in advanced disease. Standard of care for invasive bladder cancer is radical cystectomy and cisplatin-based chemotherapy. However, metastatic disease remains largely incurable and markers capable of identifying patients who are likely to respond, or not, are currently lacking. Thus, there is an urgent need to identify and characterize the molecular alterations that underlie lethal disease, and to translate this knowledge into novel strategies for BCA management. Recent profiling studies have identified discrete BCA subtypes that display distinct biological behaviors. `Basal'-type bladder cancers are characterized by increased aggressiveness, elevated EGFR pathway activation, and enhanced sensitivity to EGFR-targeted kinase inhibition. However, the mechanisms underlying EGFR activation in BCA remain unknown. New data from our group have identified SH3GL2 (endophilin A1) as a regulator of EGFR-dependent signaling in BCA. Analysis of human BCA specimens revealed a significant loss of SH3GL2 expression with disease progression. SH3GL2 silencing enhanced tumor cell survival and proliferation in vitro, and tumor growth in vivo. SH3GL2 knockdown also increased ligand-induced phosphorylation of the EGFR, SRC family kinases (SFK) and STAT3. Levels of phospho-SRC and phospho-MET were significantly enriched in bladder cancer compared to other tumor types. SH3GL2 silencing was also associated with increased membrane blebbing, a feature of the amoeboid phenotype stimulated by EGFR and MET activation that has been associated with increased tumor aggressiveness. These findings imply that inactivation of SH3GL2 is a novel upstream mechanism for activation of the EGFR, MET and their effector kinases in basal-type BCA. Based on these observations we hypothesize that loss of SH3GL2 sensitizes `basal' BCA to tyrosine kinase inhibitors (TKIs) and may serve as a biomarker of treatment sensitivity. The hypothesis will be tested with the following Specific Aims: (1) Determine the impact of SH3GL2 loss on sensitivity to EGFR and c- MET TKIs. Assays of viability, survival and particle shedding will be used to determine the biological and biochemical consequences of SH3GL2 loss on activation of EGFR/c-MET/effector pathways and sensitivity to TKIs. (2) Determine the association between loss of SH3GL2 and outcome in BCA in vivo. Quantitative multiplex imaging of tissue microarrays from BCA patients will be used to investigate the relationship between SH3GL2 expression activation of EGFR/c-MET and effectors. Orthotopic xenografts will be used to assess the impact of SH3GL2 loss on sensitivity to TKIs in vivo. At the end of the project we will understand the role of SH3GL2 in activation of the EGFR, c-MET and effectors and in sensitization of BCA to TKIs. These studies would provide a point of departure for prospective analyses aimed at determining the utility of SH3GL2 status as an early biomarker for prophylactic TKI-based targeted therapy in BCA.